Since the discovery of an acetyl group at the amino-terminus of tobacco mosaic virus coat protein, a number of N.sup..alpha. -acetylated proteins have been found in animals, plants, and their viruses, and also in bacteria and fungi. N.sup..alpha. -acetylation is therefore considered one of the typical modifications of proteins in living organisms. Moreover, in some eukaryotic cells, it has been suggested that more than 80% of the intracellular soluble proteins are N.sup..alpha. -acetylated (Brown, J. L., J. Biol. Chem. 254:1447-1449 (1979)).
The biological significance of N.sup..alpha. -acetylation of proteins is still an open question (see Tsunasawa et al., Method Enzymol. 106:165-170 (1984)). It has been proposed that this post-translational modification protects intracellular proteins from proteolysis. However, this does not hold true for all proteins. In the case of actin from slime mold, proteolytic degradation becomes slower when the protein is N.sup..alpha. -acetylated. In contrast, cat hemoglobin is degraded at the same rate irrespective of N.sup..alpha. -acetylation (Tsunasawa et al., 1984).
Recent results from DNA sequencing have shown that in structural genes for the secretory proteins that are N.sup..alpha. -acetylated, the codon for the acetylated amino-terminal residue is directly preceded by the initiation codon without the insertion of additional codons for amino acids (Tsunasawa et al., 1984). Little effort has been made to understand the relationship between N.sup..alpha. -acetylation and the transport of secretory proteins across biological membranes. To understand completely the function of N.sup..alpha. -acetylation, it will be important to identify the N.sup..alpha. -acetylated amino acids in proteins and peptides on a microanalytical scale. For this purpose, removal of the N.sup..alpha. -acetyl group or the N.sup..alpha. -acetyl amino acid must be efficiently achieved.
Acyl-Peptide Hydrolase (APH) has been successfully used for the hydrolysis of N.sup..alpha. -acylated peptides. One such enzyme, which was purified from animal liver, can liberate the N.sup..alpha. -acetyl amino acid from rather short peptides derived from N.sup..alpha. -acetylated proteins (Tsunasawa et al., 1984). The substrate specificity is broad for the amino terminal residue. APH cleaves the N.sup..alpha. -terminal acetylated or formylated amino acid from a blocked peptide (Jones et al., BBRC 126:933 (1985)). This enzyme catalyzes the hydrolysis of a diverse number of peptides and displays different pH optima for certain substrates in doing so. This enzyme may also play a pivotal role in the processing of polypeptide chains during biosynthesis. APH has been purified from rat liver (Tsunasawa et al., J. Biochem. 77:89-102 (1975)); from bovine liver (Gade et al., Biochim. Biophys. Acta 662:86-93 (1981))!; from porcine liver (Tsunasawa et al., J. Biochem. 93:1217-1220 (1983)); from rat brain (Marks et al., J. Neurochem. 41:201-208 (1983)); and from human erythrocytes (Jones et al., Biochem. and Biophys. Res. Comm. 126:933-940 (1985)).
A rat liver acyl-peptide hydrolase (APH), which catalyzes the hydrolysis of the acetylated residue from N.sup..alpha. -acetylated peptides was recently purified to homogeneity, and various inhibition experiments indicated that it was likely a serine protease, utilizing a charge relay system involving serine, histidine, and probably a carboxyl group (Kobayashi, K. and Smith, J. A., J. Biol. Chem. 262:11435-11445 (1987)). However, it is not yet clear whether acyl-peptide hydrolase is a unique serine protease.
In order to facilitate a more complete understanding of the regulation of rat acyl-peptide hydrolase in vivo, it is, therefore, desirable to clone and sequence the rat acyl-peptide hydrolase gene.